The present invention relates generally to the field of amplifying nucleic acids, more particularly to methods for producing cDNA from mRNA, sequencing DNA, and constructing cDNA libraries.
The characterization of cell specific gene expression finds application in a variety of disciplines, such as in the analysis of differential expression between different tissue types, different stages of cellular growth or between normal and diseased states, and the like. Fundamental to the characterization of cell specific gene expression is the detection of mRNA, and the construction of comprehensive cDNA libraries. However, the detection of mRNA is often complicated by one or more of the following factors: cell heterogeneity, paucity of material, or limits of low abundance mRNA detection.
In a general method of constructing cDNA libraries, polyA mRNA is prepared from the desired cells and the first strand of the cDNA is prepared from the polyA mRNA using a RNA-dependent DNA polymerase (xe2x80x9creverse transcriptasexe2x80x9d) and an oligodeoxynucleotide primer of 12 to 18 thymidine residues. In another method, the primer contains one or two nucleotides at one end that can hybridize to the mRNA sequence upstream of the polyA tail. Usually, the first polyA-non-complementary nucleotide is a deoxyadenylate, deoxyguanylate, or deoxycytidylate (xe2x80x9cdCxe2x80x9d), and the second nucleotide can be any deoxynucleotide. The use of 2 nucleotides can provide a moreaccurate positioning of the primer at the junction between mRNA and the polyA tail.
The second strand of the cDNA is synthesized by one of several methods, the more efficient of which are commonly known as xe2x80x9creplacement synthesisxe2x80x9d and xe2x80x9cprimed synthesis.xe2x80x9d Replacement synthesis involves the use of ribonuclease H (xe2x80x9cRNAase Hxe2x80x9d), which cleaves the phosphodiester backbone of RNA that is in a RNA:DNA hybrid leaving a 3xe2x80x2 hydroxyl and a 5xe2x80x2 phosphate, to produce nicks and gaps in the mRNA strand, creating a series of RNA primers that are used by E. coli DNA polymerase I, or its xe2x80x9cKlenowxe2x80x9d fragment, to synthesize the second strand of the cDNA. This reaction is very efficient; however, the cDNAs produced most often lack the 5xe2x80x2 terminus of the mRNA sequence.
Primed synthesis to generate the second cDNA strand is a general name for several methods which are more difficult than replacement synthesis yet clone the 5xe2x80x2 terminal sequences with high efficiency. In general, after the synthesis of the first cDNA strand, the 3xe2x80x2 end of the cDNA strand is extended with terminal transferase, an enzyme which adds a homopolymeric xe2x80x9ctailxe2x80x9d of deoxynucleotides, most commonly deoxycytidylate. This tail is then hybridized to a primer of oligodeoxyguanidylate or a synthetic fragment of DNA with an deoxyguanidylate tail and the second strand of the cDNA is synthesized using a DNA-dependent DNA polymerase.
Once both cDNA strands have been synthesized, the cDNA library is constructed by cloning the cDNAs into an appropriate plasmid or viral vector. In practice this can be done by directly ligating the blunt ends of the cDNAs into a vector which has been digested by a restriction endonuclease to produce blunt ends. Blunt end ligations are very inefficient, however, and this is not a common method of choice. A generally used method involves adding synthetic linkers or adapters containing restriction endonuclease recognition sequences to the ends of the cDNAs. The cDNAs can then be cloned into the desired vector at a greater efficiency.
One potential problem with the current method of constructing cDNA libraries is that the hybridization of the oligo dT primer to the polyA tail of the mRNA in the initial step is not perfect. The primer does not necessarily accurately position at the junction between the mRNA and its polyA tail. Therefore, there may be continuous stretches of T""s in addition to the T""s on the first strand primer. While this does not usually affect efficiencies in sequencing from the 5xe2x80x2 end, it severly compromises the ability to obtain accurate and successful sequencing from the 3xe2x80x2 (polyA tail) end. Thus, there exists a need for methods and procedures of cDNA synthesis and cloning.
Methods are provided for obtaining a DNA complementary to a mRNA by contacting the mRNA having a polyadenosine (polyA) tail with a primer mixture, where each primer in the mixture comprises at least 5 contiguous deoxythymidines and at least 2 independently selected non-deoxythymidine nucleotides near one end, and reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA.
Methods are also provided for obtaining a DNA complementary to a mRNA by contacting the mRNA having a polyA tail with a primer mixture, where each primer in the mixture comprises at least 10 contiguous deoxythymidines and a non-polyA-complementary region near one end, and reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA. The non-polyA-complementary region is selected from the group consisting of 3xe2x80x2-VV, 3xe2x80x2-VTV, 3xe2x80x2-VTVV, 3xe2x80x2-VTVVV, 3xe2x80x2-VTVVTV, 3xe2x80x2-VTTV, 3xe2x80x2-VTTTV, 3xe2x80x2-VVTVVV, and 3xe2x80x2-VVVVV, and combinations thereof, wherein V is deoxyadenosine, deoxycytidine, or deoxyguanosine, and the primer mixture may contain primers that are sense, anti-sense, or double stranded, and may contain a double stranded restriction enzyme sequence near the end opposite to the one containing the non-deoxythymidine nucleotides.
Methods are also provided for producing uni-directionally cloned complimentary DNA libraries from mRNA by contacting the mRNA having polyadenylated tails with a primer mixture, wherein each primer in the mixture has at least 10 contiguous deoxythymidines and at least two non-deoxythymidine nucleotides near one end and a double stranded restriction enzyme sequence at the opposite end, reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA, modifying the complementary DNA strand wherein the polyT tail is substantially removed, and amplifying the modified cDNA strand by inserting the strand into a cloning vector uni-directionally, and amplifying using a DNA polymerase.
Methods are also provided for producing uni-directionally cloned complimentary DNA libraries from mRNA by contacting the mRNA having a polyA tail with a primer mixture wherein each primer in the mixture has at least 15 contiguous deoxythymidines having a restriction enzyme site at one end and a non-polyA-complementary region near the opposite end, wherein the non-polyA-complementary region is selected from the group consisting of 3xe2x80x2-VV, 3xe2x80x2-VTV, 3xe2x80x2-VTVV, 3xe2x80x2-VTVVV, 3xe2x80x2-VTVVTV, 3xe2x80x2-VTTV, 3xe2x80x2-VTTTV, 3xe2x80x2-VVTVVV, and 3xe2x80x2-VVVVV, and combinations thereof, wherein V is deoxyadenosine, deoxycytidine, or deoxyguanosine, reverse transcribing the mRNA using a reverse transcriptase to produce a cDNA strand having a polyT tail, modifying the cDNA strand wherein the polyT tail is substantially removed, and amplifying the modified cDNA strand by inserting the strand into cloning vector uni-directionally, and amplifying using a DNA polymerase. The primer mixture may contain primers that are sense, anti-sense, or double stranded, and may contain a restriction enzyme site near the end opposite to the one containing the non-deoxythymidine nucleotides.
These and other objections, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.